1. Field of the Invention
The present invention relates to liquid formulations of human interferon-β. The formulations are characterized in that they have a pH in the weakly acidic to neutral range between 5 and 8 and that the interferon-β is highly stable in solution while retaining the molecular integrity.
2. Description of Related Art
Naturally occurring interferons are species-specific proteins, in some cases glycoproteins, which are produced by various cells of the body after induction with viruses, double-stranded RNA, other polynucleotides and antigens. Interferons exhibit a large number of biological activities such as, for example, antiviral, antiproliferative and immunomodulatory properties. At least 3 different types of human interferons have been identified; they are produced by leucocytes, lymphocytes, fibroblasts and cells of the immune system and termed α-, β- and γ-interferons. Individual types of interferons can furthermore be divided into a large number of subtypes.
Native, human interferon-β can be prepared commercially by superinduction of human fibroblast cell cultures with poly-IC followed by isolation and purification of the interferon-β by chromatographic and electrophoretic techniques. Proteins or polypeptides which exhibit properties similar to those of natural interferon-β can also be prepared by recombinant DNA technologies (EP-A-0 028 033; EP-A-0 041 313; EP-A-0 070 906; EP-A-0 287 075; Chernajovsky et al. (1984) DNA 3, 297-308; McCormick et al. (1984) Mol. Cell. Biol. 4 166-172). Recombinant human interferon-β can be produced both in eukaryotic cells (for example CHO cells) and by prokaryotic cells (for example E. coli)
The interferons in question are termed interferon-β-1a and interferon-β 1b respectively. In contrast to interferon-β 1b, interferon-β 1a is glycosylated (Goodkin (1994) Lancet 344, 1057-1060).
A prerequisite for the therapeutic use of interferon-β is that it is pharmaceutically formulated so that the protein is storage-stable over a prolonged period while retaining the molecular integrity. Interferon-β is unstable and subject to various degradation reactions. These include, in particular, the cleavage of peptide bonds, deamidation, oxidation of the methionin to methionin sulphide, disulphide exchange, and changes in the sugar side chain which even include deglycosylation.
Owing to the therapeutical benefit of interferons, a series of formulations have been developed in recent years; however, all of them exhibit certain disadvantages. U.S. Pat. No. 4,647,454 (Inter-Yeda Ltd.) describes a formulation of fibroblast interferon-β which can be stabilized by addition of polyvinylpyrrolidone (PVP) in the highly acidic range (pH 3.5). Other preferred auxiliaries are mannitol, human serum albumin and acetate buffers. The formulation is freeze-dried and stored at 4° C.
The Japanese Patent Specification 59 181 224 (Sumitomo Chemical Co.) describes an aqueous solution of interferons in which polar amino acids such as arginine, asparagine, glutamic acid, glutamine, histidine, lysine, serine and threonine and their sodium salts together with human serum albumin are employed for stabilizing the interferons.
The international Patent Application WO 95/31213 (Applied Research Systems ARS Holding) describes a liquid formulation for interferon-β which is stabilized by addition of a polyol, preferably mannitol, and a non-reducing sugar or an amino acid. The formulation furthermore comprises a buffer (acetate buffer pH 3.0 to 4.0) and human serum albumin. While formulas with a pH of between 5 and 6 showed an immediate loss in biological activity, the formulas preferred in the patent specification are sufficiently stable at pH values of 3.0 and 4.0. Moreover, the statement regarding stability only refers to the biological activity of the formulation, but not to the molecular integrity of the active ingredient.
The European Patent Application EP 0 215 658 (Cetus Corp.) describes a formulation for recombinant interferon-β in which the bioactive compound is dissolved in an aqueous medium at a pH of between 2 and 4 with addition of stabilizers such as human serum albumin or human plasma protein fractions and, if appropriate, dextrose. A further patent application of Cetus Corp. (WO 89/05 158) describes a formulation for interferon-β where either glycerin or polyethylene glycopolymers with a mean molecular weight of between 190 to [sic] 1600 daltons are employed as stabilizers at a pH of between 2 and 4. Suitable buffer components which are mentioned are glycine, phosphoric acid and citric acid.
The European Patent Application EP 0 217 645 (Cetus Corp.) describes pharmaceutical preparations with IL-2 or interferon-β which are dissolved in an excipient medium at pH 7 to 8 and stabilized with addition of sodium laurate as surfactant. In addition, SDS is also required as further ionic surfactant in order to stabilize these preparations.
The European Patent EP 0 270 799 (Cetus Oncology Corp.) describes a formulation for unglycosylated recombinant interferon-β in an inert water-based excipient medium which comprises non-ionic polymeric detergents as stabilizer.
The European Patent Application EP 0 529 300 (Rentschler Biotechnologie GmbH) describes liquid interferon-β formulations which comprise a concentration of 30 or 70 MU/ml recombinant IFN-β, sodium chloride and imidazole buffer or sodium phosphate buffer and have a pH of 7.5 (Example 3). These formulations are stable with regard to their biological activity for 4 weeks at a storage temperature of 25° C. However, the disadvantage of these compositions is that the concentration of interferon-β used (≧30 MU/ml) is too high for practical applications. Moreover, there is no mention in EP-A-0 529 300 of a reduction in the stability of liquid interferon-β formulations by addition of human serum albumin. In contrast, the addition of human serum albumin is stated as being preferred.
In addition to formulations for interferon-β, there are also described pharmaceutical dosage forms with interferon-α. The European Patent Specification 0 082 481 (Schering Corp.) discloses an aqueous formulation intended for freeze-drying which comprises human serum albumin, in addition to a phosphate buffer and glycine. Alanine is mentioned as further optional constituent. After reconstitution, the pH of the solution is between 7.0 and 7.4. A further patent application of Schering Corp. (WO 96/11018) discloses stable aqueous solutions in interferon-α which comprise chelating agents (NaEDTA or citric acid), a surfactant (Polysorbat 80), an isotonizing agent (sodium chloride) and suitable preservatives such as methylparaben, propylparaben, m-cresol or phenol, at a pH of between 4.5 and 7.1. With regard to the biological activity (standard method of inhibiting the cytopathic effect (CPE) of a virus as described by W. P. Protzman in J. Clinical Microbiology, 1985, 22, pp. 596-599), the aqueous formulations disclosed prove to be stable for 6 months at 25° C. (biological activity >90% of the initial activity). However, a determination of the protein content by HPLC carried out in parallel already shows losses in content of between 20.2 (Table 3) or 32.5% (Table 4) after 6 months at 25° C.
EP-A-0 736 303 (Hoffmann-LaRoche AG) discloses aqueous interferon-α compositions which, in addition to an interferon-α, comprise a non-ionic detergent, a buffer for setting the pH range between 4.5 and 5.5, benzyl alcohol and, if appropriate, isotonizing agents. A determination by HPLC identifies a residual content of 84.5% after storage for three months at 25° C. and a starting concentration of 18 MU interferon-α2a, while this value drops to 62.8% when the stabilizer benzyl alcohol is omitted.
EP-A-0 641 567 (Ciba Geigy AG) describes pharmaceutical compositions which comprise hybrid interferon-α and, as stabilizer, a buffer with a pH of between 3.0 and 5.0.
U.S. Pat. No. 5,358,708 (Schering Corp.) describes aqueous formulations of interferon-α which comprise methionine, histidine or mixtures of these as stabilizer. After storage of an interferon-α solution at 40° C. for two weeks, it is found that the active ingredient content has decreased by 20%.
The abovementioned formulations for interferons have shortcomings from the present-day view since, for example, an addition of human serum albumin for stabilizing proteins should be dispensed with, owing to the higher demands for safety from virus contamination by blood donors. Moreover, a number of the above-described formulations require the addition of amino acids and/or freeze-drying. However, freeze-dried products are complicated to produce and, accordingly, expensive and require an additional pass owing to the necessity of reconstitution, and this additional pass is frequently very difficult to perform, in particular for patients with a limited power of movement. A series of formulas have unphysiological pH values of below 5.0. While such values are not entirely unusual (see also S. Sweetana and N. J. Aders, Journal of Pharmaceutical Sciences and Technology, 1996, 50: 330-342), painful irritation must be expected in the case of intramuscular or subcutaneous application. While according to Sweetana and Akers the use of surfactants such as Polysorbat 80 is admissible, a series of side effects have been described, in particular in new-born and older children, which make the use of such auxiliaries questionable. A review of the toxicity of surfactants can be found in Attwood and Florence (Surfactant Systems, their Chemistry, Pharmacy and Biology, Chapman and Hall; London, 1983). The pharmacology of Polysorbat 80 is reviewed by R. K. Varma et al. (Arzneim.-Forsch./Drug Res. 35, 1985, 804-808).
On the basis of the abovementioned disadvantages, an optimal formulation for interferon-β should combine the following properties:                retaining the biological activity over the storage period,        retaining the molecular integrity of the active ingredient molecule over the storage period,        liquid formulation, no expensive freeze-drying and no additional reconstitution,        no risky auxiliaries such as human serum albumin or surfactants (detergents),        pH in the neutral to weakly acidic range.        